Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure–function relationships to bring more nanomedicines to the clinic.


Et
To a 14 mL glass vial was added copper (I) chloride (74.1 mg, 0.75 mmol, 1 eq) and a magnetic stirrer bar. Then a mixture of 7 mL chloroform/acetone (1:1, v/v) was added, followed by N,N,N',N'tetramethylethylenediamine (232 µL, 1.53 mmol, 2 eq), giving a bright green solution. The reaction mixture was bubbled with air for 5 min, yielding a dark blue solution. Then 4-ethylphenylacetylene (105 µL, 0.77 mmol, 1 eq) was added and reaction mixture was stirred for 1 h at room temperature, forming a dark grey solution. The reaction was quenched with 6 mL of hexane and filtered to remove precipitated catalyst. The filtrate was collected, dry loaded onto celite via rotary evaporation, and then purified using flash column chromatography using a gradient from hexane to 20 % ethyl acetate in hexane. The product was isolated as a light yellow powder (63 mg, 63%). This agrees well with previously reported assignments for this compound. 1,2 COOMe To a round bottom flask (50 mL) was added methyl 4-ethynylbenzoate (168.6 mg, 1.05 mmol, 1 eq), copper (I) chloride (10.6 mg, 0.107 mmol, 0.1 eq), and a magnetic stirrer bar. Then, 10 mL of dichloromethane/acetone (1:1, v/v), followed by N,N,N',N'-tetramethylethylenediamine (45 µL, 0.301 mmol, 0.3 eq) was added, which gave a green colour to the reaction mixture. The reaction mixture was bubbled with air for 15 min, then allowed to react for 17 h at room temperature open to the atmosphere. There was a large amount of yellow precipitate in the reaction flask following overnight reaction. The precipitate was dissolved in dichloromethane (15 mL) and dry loaded onto celite via rotary evaporation. The reaction mixture was dry loaded onto a silica column and eluted using a gradient from hexane to 20% ethyl acetate in hexane. The title compound was isolated as an off-white solid (130.3 mg, 77%).

NMe2
Compound NMe2 was synthesised following literature procedures. 5 To a 7 mL glass vial was added a magnetic stirrer bar, copper (I) chloride (9.9 mg, 0.100 mmol, 0.1 eq), N,N,N′,N′-tetramethylethylenediamine (45 µL, 0.301 mmol, 0.3 eq), activated 4 Å molecular sieves (1.5 g) and 4 mL of dichloromethane/acetone (1:1, v/v). The reaction mixture was stirred vigorously open to air for 10 min, then 4-ethynyl-N,N-dimethylaniline (148 µL, 0.999 mmol, 1 eq) was added. The reaction was stirred at room temperature for 1 h. The reaction mixture was then transferred to a round bottom flask with dichloromethane (50 mL) and dry loaded onto silica. The crude reaction mixture was purified using flash column chromatography using a gradient starting from hexane to 20% ethyl acetate in hexane. The title compound was isolated as a light yellow-brown solid (125.8 mg, 87%). The data agrees well with previously reported assignments for the compound. 6

NMe3
To a 7 mL glass vial was added a magnetic stirrer bar, NMe2 (50.3 mg, 0.174 mmol, 1 eq) and N,Ndimethylformamide (3 mL). Then, iodomethane (0.542 mL, 8.72 mmol, 50 eq) was added to the reaction mixture, which was stirred at room temperature for 72 h. The reaction mixture was quenched with methanol (2 mL), stirred for 30 min, then precipitated into diethyl ether (30 mL) and centrifuged (5000 rcf, 5 min) to collect the precipitate. The precipitate was washed with diethyl ether (3x 30 mL) via suspension and centrifugation, then the precipitate was dried with a gentle stream of nitrogen. The precipitate was dissolved in 5% acetonitrile in water with 0.1% formic acid and purified using Prep-HPLC using a 5-95% B gradient over 30 min. The fractions containing the desired product were lyophilised to give the title compound as a yellow solid (81.9 mg, 82%).  In a round bottom flask, 4-iodobenzenesulfonic acid (1.07 g, 3.78 mmol, 1 eq) was dissolved in thionyl chloride (3.37 g, 28.3 mmol, 7.5 eq), placed in an ice bath, then N,N-dimethylformamide (0.36 mL) was added. The reaction was allowed to warm to room temperature then was heated at 80 °C for 2 h. The reaction was fully dried in vacuo before the addition of 2,2,2-trifluoroethanol (2.88 mL, 38.1 mmol, 10 eq) and potassium carbonate (1.306 g, 9.45 mmol, 2.5 eq). The reaction was allowed to stir for 2 h at room temperature. Then, the reaction was dried in vacuo, dissolved in dichloromethane (100 mL) and washed with water (2x40 mL) and with brine (1x40 mL). The resultant organic layer was dried with anhydrous magnesium sulphate, filtered, and dried in vacuo to give the title compound white solid (1.14 g, 82%). To a vial was added 2,2,2-trifluoroethyl 4-iodobenzenesulfonate (1.14 g, 3.11 mmol, 1.0 eq), bis(triphenylphosphine)palladium(II) dichloride (0.209 g, 0.156 mmol, 0.05 eq), copper(I) iodide (0.118 g, 0.622 mmol, 0.2 eq) and a magnetic stirrer bar. The vial was sealed with a septum, then anhydrous degassed toluene (4 mL) and degassed N,N-diisopropylamine (4 mL) were added. The reaction mixture was bubbled with argon for 10 min then argon purged (triisopropylsilyl)acetylene (1.75 mL, 11.8 mmol, 2.5 eq) was added, then the reaction was heated at 70 °C for 12 h. The reaction was cooled to room temperature, diluted in hexane (50 mL) and filtered through celite. The filtrate was then dried in vacuo and the crude reaction mixture purified using flash column chromatography using a gradient from hexane with 0.1% triethylamine to 10% ethyl acetate in hexane with 0.1% triethylamine. The reaction mixture was then dissolved in toluene (30 mL), then diethyl ether (150 mL) was added. This mixture was filtered and the filtrate collected and dried in vacuo to yield the title compound as a pale yellow oil (72%, 0.94 g). To a vial was added 2,2,2-trifluoroethyl 4-((triisopropylsilyl)ethynyl)benzenesulfonate (100 mg, 0.59 mmol, 1.0 eq) and a stirrer bar. The reaction was purged with nitrogen, then anhydrous THF (1 mL) was added. The solvent was purged with nitrogen for 10 minutes, then 1 M TBAF in THF (0.72 mL, 0.29 mmol, 1.4 eq) was added. The reaction mixture was stirred for 2 h at room temperature. The reaction was dried in vacuo, then redissolved in dichloromethane (50 mL) then washed with brine (50 mL), dried with anhydrous magnesium sulphate, filtered and concentrated via rotary evaporation. The reaction mixture was purified by flash column chromatography using (1:1 (v/v) hexane/dichloromethane with 0.1% triethylamine) to yield the title compound as a colourless oil (55 mg, quantitative).

SO3
To a 7 mL glass vial was added copper (I) chloride (14.6 mg, 0.147 mmol, 0.5 eq), a magnetic stirrer bar and 2,2,2-trifluoroethyl 4-ethynylbenzenesulfonate (101.7 mg, 0.38 mmol, 1 eq). Then dichloromethane (0.5 mL) was added, followed by N,N,N',N'-tetramethylethylenediamine (85 μL, 0.567 mmol, 2 eq). After overnight stirring at room temperature in air, the crude product was dissolved in dichloromethane (3 mL) and washed with water (2x 40 mL) and brine (1x 40 mL), followed by drying over anhydrous sodium sulphate, filtration and concentration in vacuo. The precipitate was dry loaded onto silica and then eluted using a gradient from hexane with 0.1% triethylamine to 30% ethyl acetate in hexane with 0.1% triethylamine. The resultant solid was dissolved in dichloromethane (0.5 mL) and 2 M sodium hydroxide in methanol (50 μL) and stirred overnight at room temperature. The solution was dried under nitrogen then under vacuum and the product was purified by Preparative-HPLC to yield an off-white solid 2 (45 mg, 43%).

Supporting Figures
Supporting Figure S1. Additional repeats of relative Raman intensity of model cargoes. Measurements performed in DMSO with cargoes at 2 mM (peak at 2208-2225 cm -1 ) and EdU at 40 mM. Spectra normalised to EdU peak intensity (peak at 2107 cm -1 ). Spectra measured at 1 s for 10 acquisitions with 532 nm excitation laser.
Supporting Figure S2. The aim of the modelling was to understand the variation of PDMS mass, corresponding to the 708 cm -1 peak, with 1 mM initial cargo mass, corresponding to the 2208/2220 cm -1 peak. Therefore we formed a model showing the theoretical distribution between polymer and dialkyne mass if membrane or core loading was performed.
We first found the membrane volume and core volume for particles of radii, , from 25-300 nm with a 10 nm membrane thickness (Supporting Figure S9a (1) We used the number of chains per particle, , to calculate polymer mass per particle. To find we used the experimental value from static light scattering that there were ~10 4 PMOXA-b-PDMS-b-PMOXA chains in a 100 nm diameter polymersome, 8 which we could divide by our calculated for a 100 nm diameter particle to find the expected volume per chain = 2.6 x 10 -23 L. We could then find the number of chains per particle at different radii from = (3).
We then used the known polymer molecular weight and PDMS mass fraction to calculate polymer mass and PDMS mass per particle (Supporting Figure S9c).
For membrane loading, the assumptions of complete, even loading into the membrane mean that membrane and cargo will be colocalised. Therefore we used the experimental weight ratio for 1 mM loading to calculate the cargo mass, , for a given polymer mass, where increased PDMS mass was given by unilamellar vesicles of increasing radius = × (6).
For core loading we again assumed cargo from the initial feed formed a homogenous solution entirely within the core volume, with no partitioning into the membrane. We therefore calculated the cargo mass per particle from the experimental cargo concentration, , and the calculated (eqn 2) where is the cargo molar mass.
Supporting Figure S9. Theoretical modelling summary. a) Schematic showing assumed core (blue) and membrane (red) loading modes for a vesicle of radius r. b) Summary of change in core and membrane volumes for unilamellar vesicles with identical membrane thickness and increasing radius. c) Variation of PDMS mass with radius for simplified vesicle model. Main model predictions (variation of PDMS mass and model cargo mass) shown in main text.